1. Field of the Invention
This invention relates to a method of oxidatively decarboxylating a peptide, comprising combining a peptide with EpiD, wherein the peptide comprises at its carboxy terminus the amino acid sequence X.sub.1 X.sub.2 X.sub.3 X.sub.4 X.sub.5 X.sub.6 C, (SEQ ID NO:1) wherein X.sub.1, X.sub.2, X.sub.3 and X.sub.4 are any one of the twenty common amino acids, X.sub.5 is Tyr, Val, Met, Phe, Leu, Ile or Trp, and X.sub.6 is Cys, Ala, Ser, Val, or Thr, with the proviso that the carboxy terminus of said peptide is not SFNSYCC, (SEQ ID NO:2) SFNSFCC, (SEQ ID NO:3) SFNSWCC (SEQ ID NO:4) or SFNSYSC, (SEQ ID NO:5) whereby the oxidative decarboxylation of the peptide occurs.
2. Related Art
Some polypeptide antibiotics such as nisin, subtilin, duramycin, cinnamycin, ancovenin, Ro 09-0198 and epidermin contain dehydroamino acids and lanthionine bridges. These polypeptides are produced by various respective strains of microorganism. Nisin for example can be produced by cultivating strains of Streptococcus lactin, and subtilin by cultivation of Bacillus subtilis.
The genetic basis for the biosynthesis of these antibiotics has not, hitherto, been elucidated. Thus, it has not been known, for example, whether biosynthesis of such antibiotics and, in particular, the formation of the unusual amino acids found therein occurs via ribosomal synthesis or via multi-enzyme complexes.
It addition it was not known whether the precursor proteins of such antibiotics were coded by distinct structural genes or were the degradation products of larger proteins.
In the course of work carried out to establish the structural gene of epidermine, it has been established that surprisingly the above mentioned antibiotics, in particular epidermin, are each coded by a distinct structural gene, and that processing of a presequence polypeptide is carried out by an enzymatic complex which effects formation of dehydroamino residues and/or thioether bridges.
Furthermore, the multi-enzyme complex may be involved in the secretion of the protein through the cell membrane into the culture supernatant, as well as processing a prepolypeptide. In this connection, such activity may be associated with a pre-sequence possessed by the pre-polypeptide, e.g., as in the case of the -30 to -1 sequence of pre-epidermin as described below.
It has unexpectedly been determined that the multienzyme complex responsible for the posttranslational modification of pre-epidermin is located on the 54 kb plasmid pTu32 of Staphylococcus epidermis Tu 3298/DSM 3095.
The six genes (ORFs) responsible for the production of epidermin are designated herein epi A, B, C, D, Q and P and are clustered within 8 kb and the proteins for which they code are designated Epi A, B, C, D, Q and P respectively; epi A encodes the 52 amino acid-long pre-epidermin. As described below, epi B, C and D are involved in the four enzymatic modification reactions (i) water elimination by a serine/threonine dehydratase, (ii) sulfur addition by a lanthinonine synthase, (iii) C-terminal decarboxylation by a cysteine decarboxylase and (iv) double bond formation. Epi P protein is believed to be responsible for cleaving the mature epidermin from the N-terminal leader peptide, based on its striking homologies with the essential domain of serine proteases (Koide et al., J. Bacteriol. 167:110-116 (1986); Meloun et al., FEBS Lett. 183:195-200 (1985); and Stahl et al., J. Bacteriol. 158:411-418 (1984)) while Epi Q is believed to be a regulatory protein regulating epidermin biosynthesis, based on its distinct homology to the pho B gene of E. coli (Makino et al., J. Mol. Biol. 190:37-44 (1986)), the fact that both proteins are of a similar size with 205 (epi Q) and 229 (pho B) amino acid residues, the observed homology of 24.2% extending over the 153 C-terminal amino acid residues and the hydrophilicity plots of both proteins.
The enzyme EpiD has been purified and identified as a flavoprotein with flavin mononucleotide as coenzyme (Kupke, T. et al., J. Bacteriol. 174:5354-5361 (1992)). The EpiD* gene of the epi-mutant S. epidermis TUS3298/EMS 11 has been expressed as a maltose-binding protein (MBP)-EpiD* fusion protein in Escherichia coli. Unlike MBP-EpiD, this fusion protein MBP-EpiD* cannot bind the flavin coenzyme. DNA sequencing of EpiD* identified a point mutation that led to replacement of Gly.sup.93 with Asp (Kupke, T. et al., J. Bacteriol. 174:5354-5361 (1992)).
The substrate peptide EpiA and the mutated peptide EpiAR-1Q have been purified by factor Xa cleavage from MBP fusions. The identity of purified EpiA and EpiAR-1Q have been confirmed by electrospray mass spectrometry (ES-MS) and amino acid sequencing (Kupke, T., et al., FEMS Lett 112:43-48 (1993)). EpiA consists of an NH.sub.2 -terminal leader peptide (amino acids -30 to -1 ) and a COOH-terminal proepidermin (amino acids +1 to +22) (Schnell, N., et al, Nature 333:276-278 (1988)). The last two amino acids of EpiA are cysteine residues.
Recently, it was demonstrated that under reducing conditions, EpiD reacts with unmodified precursor peptides EpiA and EpiAR-1Q and with the COOH-terminal proepidermin fragment of EpiA as shown by reversed phase chromatography and ES-MS (Kupke, T., et al., J. Biol. Chem. 269:5653-5659 (1994)). A decrease in mass by 46 Da was observed, and an increase in absorbance at 260 nm of the modified peptides. Sequence analysis of modified proepidermin indicates that one of the two last cysteine residues of proepidermin is modified by EpiD. A model has been proposed that EpiD catalyzes the removal of two reducing equivalents from the side chain of the COOH-terminal cysteine residue (Kupke, T., et al., J. Biol. Chem. 269:5653-5659 (1994)). A double bond is formed, and the flavin mononucleotide coenzyme is reduced. The COOH-terminal carboxyl group is then removed by a decarboxylation reaction resulting in the COOH-terminal enethiol side chain. The oxidated and decarboxylated peptide is unstable and is nonenzymatically converted to less hydrophobic peptides. The reaction is inhibited by Zn.sup.2+, and the oxidative decarboxylated peptide is probably stabilized by Zn.sup.2+ (Kupke, T., et al, J. Biol Chem. 269:5653-5659 (1994)). It was concluded that the oxidoreductase EpiD is involved in formation of the COOH-terminal S-(Z)-2-aminovinyl!-D-cysteine.
Clearly, further characterization of this novel posttranslational modification reaction is necessary. One major question regarding posttranslational modifications concerns the specificity of the processing reactions in selecting only a few or sometimes even only one residue for modification (Yan, S. C. B., et al., Trends Biochem. Sci. 14:264-268 (1989)).